1. Field of the Invention
The present invention relates to cancer detection, specifically a methylation specific polymerase chain reaction method for detecting the presence of gene-specific promoter methylation in tumor cells, and more specifically a nested, two-stage polymerase chain reaction method for amplifying the gene that may be altered in a particular cancer, thereby permitting cancer detection and monitoring by detecting gene inactivation in biological fluids such as sputum and blood. The invention may also find application in the field of pharmacogenomics.
2. Background Art
Cancer is one of the leading causes of death in the U.S. The high mortality from this disease stems from the inability in many cases to detect the cancer at a stage where it can be removed surgically and has not metastasized to other sites in the body. Current radio- and chemotherapy often result in disease remission, but actual cures (long-term term survival) are rare for advanced stage cancer. However, 5-year survival for cancers that are surgically removed with no evidence of distant disease is 60% to 80%. The development of biomarkers usable for early detection, to follow therapy, and to predict outcome of the disease, could greatly aid in managing cancer and increasing long-term survival.
Chemoprevention studies in current and former cancer-free subjects have largely been focused on the effect of defined interventions on cytologic and genetic changes in bronchial epithelium obtained through biopsy. This approach is predicated on the fact that virtually the entire lower respiratory tract is exposed to inhaled carcinogens within cigarette smoke. The resulting “field cancerization” involves the generation of multiple, independently initiated sites throughout the lungs of persons with a long history of smoking. Cytological and genetic changes have been detected throughout the bronchial tree. Many of the genetic changes detected in the bronchial epithelium may have limited utility for assessing lung cancer risk and response to intervention therapy. For example, assays for loss of heterozygosity (LOH) in cytologically normal tissue have limited sensitivity, and quite often subjects will not be informative for the microsatellite marker used to assess LOH at a specific chromosome locus. Moreover, some LOH changes appear less frequently in former smokers compared to current smokers, suggesting they may be associated more with exposure. In addition, the conduct of large population-based prevention trials will necessitate the use of biological fluids such as sputum that can be obtained non-invasively for monitoring drug response. Neither polymerase chain reaction-nor fluorescent in situ hybridization (FISH)-based assays have the sensitivity to detect LOH in sputum. At present, there is no extensive data on methylation in the bronchial epithelium in either persons with prevalent lung cancer or cancer-free smokers. Still further, the effect of smoking cessation on these markers has not been evaluated.
The development of cancer involves inactivation of many different types of genes in a cell. It is this inactivation that is largely responsible for a normal cell becoming a tumor cell. One type of gene inactivation that occurs frequently in cancer is called CpG island methylation of gene promoters. This process involves a change in the portion of the gene, the promoter, which is responsible for maintaining its activity. CpG island methylation is responsible for inactivating many different cellular genes.
Because current and even former cigarette smokers have increased bronchial secretions that contain exfoliated cells from the bronchial tree, the analysis of sputum from these individuals has been an area of research for marker development. The late Dr. Geno Saccomanno demonstrated that premalignant cytologic changes can be detected several years prior to a clinical diagnosis of lung cancer in high-risk subjects. Unfortunately, these studies were difficult to replicate, most likely because of the skills required for identifying subtle nuclear changes in cells that often comprise <5% of the sputum slide. Subsequent studies suggested that molecular assays could be used to enhance the predictive value of sputum samples. Mutations within the K-ras gene have been detected in sputum specimens collected prior to tumor resection; identical microsatellite alterations have been detected in primary tumors and corresponding sputum samples. However, the known methods to detect both alterations lack sensitivity, and the overall prevalence of these changes in non-small cell lung cancer (NSCLC) is less than 25%.
Recently, Drs. James Herman and Stephen Baylin developed a technique, called methylation specific polymerase chain reaction (MSP), to detect the presence of gene-specific promoter methylation in tumor cells. This technique is described in U.S. Pat. No. 5,786,146 to Baylin et al., and U.S. Pat. No. 6,017,704 to Herman, et al., the teachings of which are incorporated herein by reference. With the MSP approach, it is possible to detect one copy of a specific methylated gene in the background of 1,000 unmethylated gene copies. This approach is useful for detection of specific methylated genes in primary tumors, but has extremely limited utility for detecting gene inactivation in biological fluids such as sputum, plasma, urine, and fecal stool. Evaluating biological fluids is the most noninvasive and economical approach for population-based screening. Because these fluids contain DNA or cells that are mainly normal cells, increased sensitivity of the MSP procedure is essential for detecting the presence of the tumor cells.
Against the foregoing background, the present invention was developed.